The present invention relates generally to plant molecular biology. In particular, it relates to nucleic acids and methods for improving glutenin content of plants.
The glutenins, which include both high molecular weight (HMW) glutenin subunits and low molecular weight (LMW) glutenin subunits, comprise an economically important class of wheat seed storage proteins. The apparent molecular weights of the individual HMW glutenin polypeptides or subunits range from 90 to 200 kDa. These subunits crosslink by disulfide bonds among themselves and with LMW glutenin polypeptides to form polymers exceeding one million daltons in molecular weight. HMW glutenins constitute 8-10%, while LMW glutenins constitute 15-20% of the total endosperm protein. Both HMW and LMW glutenin proteins play important functional roles in determining the end-uses of wheat flour.
In wheat, HMW glutenins are encoded at the Glu-1 loci on the long arms of the group 1 chromosomes. Each locus consists of two separate genes, encoding an x-type and a y-type subunit, respectively. These pairs have never been confirmed to be separated by recombination. This has made determination of their separate contributions to bread dough properties difficult to assess by genetic correlation studies. For a review of the genetics and biochemistry of glutenin polypeptides, see, Shewry et al., J. Cereal Sci. 15:105-120 (1992).
Both the quantity and identity of specific HMW glutenin alleles contribute to the differences in bread-making quality of various cultivars. For instance, deletion of glutenin genes results in a decrease in the overall levels of HMW glutenins, which results in decreases in bread-making quality (see, e.g., Lawrence et al. J. Cereal Sci 7:109-112 (1988)).
The effects of overproducing HMW glutenin on protein accumulation and baking quality has not been assessed because such lines of wheat have not been found among natural populations. In addition, direct alteration of the glutenin subunits that form the polymers is not possible using standard breeding methods. Thus, the art lacks reproducible and efficient methods of producing lines with altered glutenin contents. The present invention addresses these and other needs.
The present invention provides methods of increasing glutenin in the endosperm of wheat plants. The methods and plants of the invention therefore are useful in providing flour and dough having improved end-use properties. The methods comprise introducing into a parental wheat plant a recombinant expression cassette comprising a nucleic acid encoding a glutenin polypeptide and selecting progeny wheat plant having increased glutenin content in the endosperm of mature seed. The glutenin content of the progeny is preferably at least about 15% greater than the glutenin of the parental wheat plant.
Any method of introducing the expression cassette into the parental plant can be used. Particle bombardment is a convenient method for producing transgenic plants. Once the expression cassette is stably integrated into the genome, standard sexual crosses can be used to introduce the expression cassette into desired lines.
In some embodiments, the nucleic acid introduced into the plant encodes a chimeric glutenin polypeptide. For instance, the chimeric polypeptide may comprise sequences from an x-type glutenin polypeptide and a y-type glutenin polypeptide. Exemplary genes for this purpose include the Glu-D1-1b gene and the Glu-D1-2b gene.
The expression cassette may further comprise a seed-specific promoter to direct expression of the introduced nucleic acid to the endosperm. A convenient promoter for this purpose is the promoter from the Glu-D1-2b gene.
Any wheat cultivar can be used as the parental line in the present invention. An exemplary cultivar is Bobwhite.
The invention also provides wheat plants comprising a recombinant expression cassette comprising a nucleic acid encoding a glutenin polypeptide. The plants of the invention have a glutenin content in the endosperm of a mature seed at least about 15% greater than the glutenin content in the endosperm of a mature seed from a parental wheat plant. The percentage of total endosperm protein which is glutenin will usually depend upon the glutenin content of the parental line. Usually, HMW glutenins account for at least about 15% of the total protein in the endosperm of the mature seed from plants of the invention.
The invention further provides recombinant constructs comprising a wheat glutenin gene promoter of about 400 to about 2800 nucleotides operably linked to a heterologous polynucleotide sequence. These constructs are particularly useful in directing expression of the heterologous sequence to seeds of transgenic plants. An exemplary wheat glutenin gene from which the promoters of the invention can be derived is Glu-D1-2b.
Definitions
The term xe2x80x9cplantxe2x80x9d includes whole plants, plant organs (e.g., leaves, stems, roots, flowers, etc.), seeds and plant cells and progeny of same. The class of plants which can be used in the methods of the invention is generally as broad as the class of higher plants amenable to transformation techniques, including both monocotyledonous and dicotyledonous plants.
A xe2x80x9cheterologous sequencexe2x80x9d is one that originates from a foreign species, or, if from the same species, is substantially modified from its original form. For example, a promoter operably linked to a heterologous structural gene is from a species different from that from which the structural gene was derived, or, if from the same species, one or both are substantially modified from their original form.
A xe2x80x9cglutenin polypeptidexe2x80x9d is a gene product of a glutenin gene or glutenin polynucleotide sequence. A glutenin polypeptide can be either a LMW glutenin or a HMW glutenin. A glutenin polypeptide contains cysteine residues by which disulfide bonds are formed with other glutenin polypeptides to form polymers. The composition and size of the repeat region is also important to polymer formation.
A xe2x80x9cchimeric glutenin polypeptidexe2x80x9d is glutenin gene product that comprises a modified amino acid sequence. Modifications, as explained in detail below, can be in the form of substitutions, deletions, or additions of single amino acids or groups of amino acids. Thus, chimeric glutenin polypeptides can be hybrid glutenin polypeptides comprising sequences from two or more different subunits or may be polypeptides in which single amino acid modifications are made.
In the case where an inserted polynucleotide sequence is transcribed and translated to produce a functional glutenin polypeptide, one of skill will recognize that because of codon degeneracy, a number of polynucleotide sequences will encode the same polypeptide. These variants are specifically covered by the terms xe2x80x9cglutenin genexe2x80x9d or xe2x80x9cglutenin polynucleotide sequencexe2x80x9d. In addition, the terms specifically include those full length sequences substantially identical (determined as described below) with a glutenin gene sequence and that encode proteins that retain the function of the glutenin polypeptide. Thus, in the case of wheat glutenin genes disclosed here, the term includes variant polynucleotide sequences which have substantial identity with the sequences disclosed here and which encode glutenin polypeptides capable of crosslinking by disulfide bonds with other glutenin polypeptides to form glutenin polymers.
Two polynucleotides or polypeptides are said to be xe2x80x9cidenticalxe2x80x9d if the sequence of nucleotides or amino acid residues, respectively, in the two sequences is the same when aligned for maximum correspondence as described below. The term xe2x80x9ccomplementary toxe2x80x9d is used herein to mean that the complementary sequence is identical to all or a portion of a reference polynucleotide sequence.
Sequence comparisons between two (or more) polynucleotides or polypeptides are typically performed by comparing sequences of the two sequences over a segment or xe2x80x9ccomparison windowxe2x80x9d to identify and compare local regions of sequence similarity. Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman and Wunsch J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson and Lipman Proc. Natl. Acad. Sci. (U.S.A.) 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis., or by inspection.
xe2x80x9cPercentage of sequence identityxe2x80x9d is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.
The term xe2x80x9csubstantial identityxe2x80x9d of polynucleotide sequences means that a polynucleotide comprises a sequence that has at least 60% sequence identity, preferably at least 80%, more preferably at least 90% and most preferably at least 95%, compared to a reference sequence using the programs described above using standard parameters. One of skill will recognize that these values can be appropriately adjusted to determine corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning and the like. Substantial identity of amino acid sequences for these purposes normally means sequence identity of at least 40%, preferably at least 60%, more preferably at least 90%, and most preferably at least 95%. Polypeptides which are xe2x80x9csubstantially similarxe2x80x9d share sequences as noted above except that residue positions which are not identical may differ by conservative amino acid changes. Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains is cysteine and methionine. Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine.
Another indication that nucleotide sequences are substantially identical is if two molecules hybridize to each other under stringent conditions. Stringent conditions are sequence dependent and will be different in different circumstances. Generally, stringent conditions are selected to be about 5xc2x0 C. to about 20xc2x0 C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. However, nucleic acids which do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides which they encode are substantially identical. This may occur, e.g., when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code.